Targeting Reactive Carbonyls for Identifying Natural Products and Their Biosynthetic Origins

Abstract

Natural products serve important roles as drug candidates and as tools for chemical biology. However, traditional natural product discovery, largely based on bioassay-guided approaches, is biased towards abundant compounds and rediscovery rates are high. Orthogonal methods to facilitate discovery of new natural products are thus needed, and herein we describe an isotope tag-based expansion of reactivity-based natural product screening to address these shortcomings. Reactivity-based screening is a directed discovery approach in which a specific reactive handle on the natural product is targeted by a chemoselective probe to enable its detection by mass spectrometry. In this study, we have developed an aminooxy-containing probe to guide the discovery of aldehyde- and ketone-containing natural products. To facilitate the detection of labeling events, the probe was dibrominated, imparting a unique isotopic signature to distinguish labeled metabolites from spectral noise. As a proof of concept, the probe was then utilized to screen a collection of bacterial extracts, leading to the identification of a new analog of antipain, deimino-antipain. The bacterial producer of deimino-antipain was sequenced and the responsible biosynthetic gene cluster was identified by bioinformatic analysis and heterologous expression. These data reveal the previously undetermined genetic basis for a well-known family of aldehyde-containing, peptidic protease inhibitors, including antipain, chymostatin, leupeptin, elastatinal, and microbial alkaline protease inhibitor (MAPI), which have been widely used for over 40 years.

Toward the Dark Matter of Natural Products

“…Recently, by taking advantage of the reactivities of the constituents, the chemo‐diversity of natural products has been expanded by the direct application of chemical reactions to extracts of Chinese traditional medicines . Similarly, reactivity‐based screening using probes targeting specific functional groups has also emerged as a natural product discovery tool . These new methods are quite suitable for the mining of reactive natural products.…”

Reclassification of Streptomyces hygroscopicus subsp. glebosus and Streptomyces libani subsp. rufus as later heterotypic synonyms of Streptomyces platensis

“…Abundant growth occurs on ISP 2, 3, 4 and 5 agars. White or off-white to dark grey aerial spore mass is formed on light yellow to yellow substrate hyphae on [28]; lydicamycin, pks/ nrps for lydicamycin and TPU-0037-A to -D [29,30]; oxy, t2pks for oxytetracycline [31]; oxz, pks/nrps for oxazolomycin [32]; vem, t3pks for venemycins [33]; scaP, t1pks for caniferolides [34]; may, t2pks for mayamycin [35]. Gene clusters whose products can be predicted by bioinformatic analysis are boldfaced and the others such as t1pks, t2pks, t3pks, nrps and pks/nrps are orphans.…”

Reactivity-based screening for citrulline-containing natural products reveals a family of bacterial peptidyl arginine deiminases

“…RBS has enabled the discovery and subsequent characterization of RiPPs, non-ribosomal peptides, polyketides, and hybrids thereof (e.g. cyclothiazomycin C, 31 deimino-antipain, 32 hygrobafilomycin JBIR-100, 33 and tirandalydigin, 34 etc.). RBS is therefore a valuable tool for bridging bioinformatics-guided NP identification with NP production.…”

“…Additionally, an RBS-based NP discovery strategy permits rapid dereplication of already-characterized NPs, 31 detection of low-abundance species using sensitive MS-based techniques such as matrix-assisted laser/desorption ionization time-of-flight MS (MALDI-TOF-MS), and can employ variable probe chemistries that enhance NP detection due to unique isotopic patterns of labeled compounds or selective enrichment through affinity purification. 32,35,36 Herein, we describe the expansion of RBS as a tool to aid in the discovery of the PAD responsible for deimination activity on the citrulassin family of lasso peptides. Repurposing the work of Thompson, 37, 29 a phenylglyoxal probe was employed to specifically label primary ureido groups over primary guanidino groups.…”